HIV Type 1 Nef is released from infected cells in CD45(+) microvesicles and is present in the plasma of HIV-infected individuals

Abstract

HIV-1 Nef has been demonstrated to be integral for viral persistence, infectivity, and the acceleration of disease pathogenesis (AIDS) in humans. Nef has also been detected in the plasma of HIV-infected individuals and is released from infected cells. The form in which Nef is released from infected cells is unknown. However, Nef is a myristoylated protein and has been shown to interact with the intracellular vesicular trafficking network. Here we show that Nef is released in CD45-containing microvesicles. This microvesicular Nef (mvNef) is detected in the plasma of HIV-infected individuals at relatively high concentrations (10 ng/ml). It is also present in tissue culture supernatants of Jurkat cells infected with HIV(MN). Interestingly, plasma mvNef levels in HIV(+) patients did not significantly correlate with viral load or CD4 count. Microvesicular Nef levels persisted in the plasma of HIV-infected individuals despite the use of antiretroviral therapy, even in individuals with undetectable viral loads. Using cell lines, we found Nef microvesicles induce apoptosis in Jurkat T-lymphocytes but had no observed effect on the U937 monocytic cell line. Given the large amount of mvNef present in the plasma of HIV-infected individuals, the apoptotic effect of mvNef on T cells, and the observed functions of extracellular soluble Nef in vitro, it seems likely that in vivo mvNef may play a significant role in the pathogenesis of AIDS.

FIG. 1.

Nef expression and HIV-1 infection induce vesicle formation. TEM image of conditioned media from HEK293 cells transfected with (A) Nef expression plasmid or (B) mock-transfected. Conditioned media collected 3 days posttransfection and subjected to ultracentrifugation (400,000 × g). Pellets were fixed, embedded, and stained with 2% uranyl acetate. Samples were visualized using a JEOL 1200 EX transmission microscope. (C) Culture supernatant from HEK293 transfected with a Nef expression plasmid was subjected high-speed differential centrifugation. Supernatants were centrifuged for 10 min at 300 × g, followed by 30 min at 10,000 × g, and then centrifuged for 1 h at 300,000 × g. Nef was detected in vesicles pelleted at 300,000 by immunoblot using anti-Nef monoclonal. S, supernatants; P, pellet.

FIG. 2.

Nef vesicles captured using anti-CD45 magnetic bead separation. Supernatants from HIV-1-infected cells were harvested 15 days postinfection and vesicles/virions were concentrated by centrifugation at 200,000 × g for 1 h on a 20% sucrose cushion. The pellet was resuspended in 1 × TNE buffer and subjected to CD45 magnetic bead separation. Each fraction was collected, ultracentrifuged at 400,000 × g for 1 h, and then separated by SDS-PAGE. Fractions (SM = starting material, FT = flow through, W = wash, and E = eluate) were analyzed by Western blot for HIV proteins using anti-HIV sera (upper panel). The singular band present in the eluate was confirmed to be Nef via an immunoblot using an anti-Nef monoclonal antibody (lower panel). Nef⁺, recombinant Nef protein.

FIG. 3.

Subtilisin treatment reduces CD45 capture of Nef microvesicles capture. Nef microvesicles from supernatants of HIV-infected Jurkat cells (15 days postinfection) were isolated as described in Fig. 2, treated for 4 h with subtilisin (2 mg/ml), and then CD45 affinity purified. Nef contents in subsequent SM, FT, W, and E fractions were measured by ELISA. SM = starting material, FT = flow through, W = wash, and E = eluate.

FIG. 4.

Production of CD45⁺ Nef microvesicles is cell-type dependent. Concentrations of p24 (A) microvesicles and Nef (B) were measured by ELISA in culture supernatants and CD45 captured microvesicles of HIV-1-infected Jurkat T-lymphocytes, THP-1 monocytes, and PMA-treated THP-1 (macrophage-like). T-lymphocytes release the most HIV-1 virus as measured by p24 ELISA whereas macrophage-like THP-1 releases the most Nef microvesicles.

FIG. 5.

Nef microvesicles are detected in plasma of HIV-infected subjects. Plasma was collected from uninfected (n = 15) and HIV⁺-infected individuals (n = 10). Microvesicles were ultracentrifuged (400,000 × g); pellets were resuspended in PBS and assayed for Nef using a Nef-capture ELISA. The median value of Nef in plasma is 10.3 (ng/1 ml plasma). Statistical significance was determined using a two-tailed Mann–Whitney analysis. ***p-value < 0.001.

FIG. 6.

Nef microvesicles in plasma of HIV-infected subjects are isolated by CD45 affinity. CD45⁺ microvesicles in plasma of (A) uninfected and (B) HIV⁺ donors were isolated as described previously in Fig. 2. Fractions generated during CD45 affinity (SM, FT, W, and E) were ultracentrifuged (400,000 × g); the pellets were resuspended in PBS and assayed for Nef via Nef capture ELISA. Nef was detected in the SM and CD45 captured material (E). SM = starting material, FT = flow through, W = wash, and E = CD45 captured eluate.

FIG. 7.

Exposure to vesicular Nef induces apoptosis. Caspase-3 activation in Jurkat and U937 monocytic cells exposed to Nef for 3, 6, and 18 h was measured using a 3-plex Luminex kit. Statistical significance was determined using ANOVA, *p-value <0.05.

FIG. 8.

Model of Nef microvesicle in HIV pathogenesis. HIV-infected cells release both virions and Nef microvesicles. Depending on the cell type of the microvesicle, the virion ratio varies. In step 1 the infected cells release both virions and Nef microvesicles. Cells (both T cells and monocytes) that come in contact with virions become infected (step 2). T cells encountering only Nef microvesicles undergo apoptosis whereas monocytes/macrophages do not (step 3). In this model Nef microvesicles are integral to HIV pathogenesis.